PROJECT SUMMARY/ABSTRACT Conventional definitions of sudden cardiac death (SCD) presume cardiac cause.4 Our NHLBI-funded ongoing POST SCD Study, which has autopsied 97% of >1000 consecutive SCDs since 2011, is the first and only prospective unselected adult SCD cohort, capturing the entirety of SCDs in a community, to use autopsy to refine SCD to true cardiac causes.5 Recent SCD risk6 and genetic studies7 have reported inconsistent results likely due to reliance on the presumed SCD phenotype. Guidelines8 and recent studies3 support postmortem targeted cardiovascular disease (CVD) genetic testing of autopsy-negative sudden deaths < 35 years, but no guidelines exist for postmortem genetic testing of older SCDs (> 35 years), nor SCDs with structural pathology. In POST SCD, 98% of cases have common pathology such as coronary artery disease (CAD), left ventricular hypertrophy (LVH), or dilated cardiomyopathy (DCM). The contribution of clinically actionable monogenic CVD has not been studied in autopsy-defined adult SCD with a background of such common cardiac pathology. Our pilot data demonstrate the potential to elucidate associations of established, pathogenic structural gene variants with arrhythmic risk to inform postmortem testing guidelines and extend and refine phenotypes. We have successfully engaged the large majority of POST SCD families to deploy a novel, highly efficient web-based tool to address a major unmet need in SCD prevention ? identifying co-segregating first degree relatives of SCD victims who are at highest risk. Finally, one of the key barriers to new approaches in SCD prevention is a lack of understanding of the acute cellular milieu that predisposes to fatal arrhythmias in the context of acquired or inherited structural pathology, e.g., LVH, fibrosis, dilation, or scar. We propose an innovative method to interrogate postmortem myocardial RNA transcripts ? reflecting the sum total effect of all possible factors in the hours prior to SCD: genetic, epigenetic, ischemia, neurohumoral, and underlying myocardial pathology ? as an insight into the acute cellular alterations that create vulnerable substrate for fatal arrhythmia. We will leverage family consent, blood, and left ventricular samples from our autopsy-defined SCDs and matched controls to address the following Specific Aims: (1A) Determine the yield of pathogenic variants in clinically actionable CVD genes in adult SCDs with autopsy sub-phenotypes; (1B) Refine genotype-phenotype correlations with pathogenic variants in HCM genes for SCDs with LVH, DCM genes for SCDs with dilated hearts, and channelopathy genes in all SCDs; (2) Elucidate genotype-phenotype correlation to identify first degree relatives with co-segregating monogenic CVD predisposing to SCD; (3) Characterize the myocardial RNA profile specific to fatal arrhythmias in the setting of sub-phenotypes of SCD. We anticipate that this project will inform guidelines for postmortem genetic testing in all adult SCDs, refine precision genotype-phenotype correlations in population SCD, filter variants of uncertain significance to potentially causative ones, and offer insights into the acute cellular milieu vulnerable to SCD.